Drug testing system with bio-artificial liver

ABSTRACT

A drug testing system using a liver-slice culture apparatus. The apparatus has a chamber with plasma and gas valves, animal liver slices being positioned securely in the chamber so as to maximize the surface area of liver slices exposed to the culture medium. Plasma is supplied to the chamber so that it rises to contact the liver slices, and is alternately removed from contacting the liver slices. Gas is supplied to the top of the chamber. The system also includes a reservoir for containing media entering and exiting the chamber. Methods are provided for assessing the toxicity of a drug or drug candidate.

BACKROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a drug testing system with a biologicalartificial liver and, more particularly, a bioreactor for evaluation,detection and testing of drug candidates, drugs and drug metabolites.

2. Discussion of Related Art

In 2001, the average cost to develop a new drug exceeded $800 million,according to a study by the Tufts Center for the Study of DrugDevelopment. Of this, approximately $16 million on average per companywas used for pre-clinical research. Reduction of testing time and costin drug development is therefore a critical factor to the survival ofmost pharmaceutical companies. In addition, since there is usually morethan one company competing in the same drug arena, any competitiveadvantage would be welcome. A major portion of drug development costs isborne during the FDA approval process. However, much of this cost cannotbe managed in the same way that pre-clinical costs can. To addresssoaring pre-clinical costs, more efficient, affordable, and timelymethods of in vivo and in vitro testing and selection of potential newdrug candidates are of significant interest in the industry.

In developing a new drug, toxicity is always an important consideration.Since the liver metabolizes most drugs, liver damage is of greatconcern. Conventional in vivo and in vitro tests utilizing small animalsand cell culture techniques are therefore widely used to assess liverfunction in drug development. However, these conventional tests haveparticular disadvantages, such as individual variation, high costs touse large animals, and loss of naturally existing characteristics ofliver in situ.

To overcome these disadvantages, cell culture systems have also beenused. However, with these models cell-to-cell connective interactionscannot be maintained for a desired length of time. This leads to failureof the testing scheme.

Bioartificial liver devices are currently being developed. It isbelieved that hepatic function can only be replaced with the biologicalsubstrate, that is, liver cells or a whole liver specimen, whichrequires the availability of liver tissue from xenogenic or humansources. Recent efforts have combined mechanical and biologic supportsystems in hybrid liver support devices. The mechanical component ofthese hybrid devices serves both to remove toxins and to create abarrier between the patient's serum and the biologic component of theliver support device. The biologic component of these hybrid liversupport devices may consist of liver slices, granulated liver, orhepatocytes from low-grade tumor cells or porcine hepatocytes. Thesebiologic components are housed within chambers often referred to asbioreactors. However problems remain with respect to maintaining thefunctionality of the individual cell lines used in these devices. Mostdevices use immortalized cell lines. It has been found that over timethese cells lose specific functions.

There are several groups developing bioartificial liver devices, forexample, Circe Biomedical (Lexington, Mass.), Vitagen (La Jolla,Calif.), Excorp Medical (Oakdale, Minn.), and Algenix (Shoreview,Minn.). The Circe Biomedical device integrates viable liver cells withbiocompatible membranes into an extracorporeal, bioartificial liverassist system. Vitagen's ELAD® (Extracorporeal Liver Assist Device)Artificial Liver is a two-chambered hollow-fiber cartridge containing acultured human liver cell line (C3A). The cartridge contains asemipermeable membrane with a characterized molecular weight cutoff.This membrane allows for physical compartmentalization of the culturedhuman cell line and the patient's ultrafiltrate. Algenix provides asystem in which an external liver support system uses porcine livercells. Individual porcine hepatocytes pass through a membrane to processthe human blood cells. Excorp Medical's device contains a hollow fibermembrane (with 100 kDa cutoff) bioreactor that separates the patient'sblood from approximately 100 grams of primary porcine hepatocytes thathave been harvested from purpose-raised, pathogen-free pigs. Bloodpasses though a cylinder filled with hollow polymer fibers and asuspension containing billions of pig liver cells. The fibers act as abarrier to prevent proteins and cell byproducts of the pig cells fromdirectly contacting the patient's blood but allow the necessary contactbetween the cells so that the toxins in the blood can be removed.

Various aspects of these devices represent improvements overpre-existing technology, but they still have particular disadvantages.The effectiveness of these devices, all of which use individualhepatocytes, is limited due to the lack of cell-to-cell interactions,which characterize the liver in its in vivo state. Accordingly, abioartificial liver with improved efficiency, viability, andfunctionality for use in drug development would be beneficial.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a system to testthe toxicity of a potential drug candidate and its metabolites.

The present invention provides a system for testing a potential drugcandidate for toxicity. The system has a liver-slice culture apparatusmade up of a chamber having a medium inlet and a gas valve, a pluralityof animal liver slices positioned securely within the chamber so as tomaximize the surface area of the liver slices exposed to the medium,means for selectively supplying and removing a medium in the chamber sothat the medium in the chamber comes into contact with the liver slices,and is removed from contact with the liver slices, and a reservoir forcontaining the medium as it enters and exits the chamber. The animalliver slices are cultured in an environment of an oxygenated gas underthe supply of the medium at regular intervals so that said slices areexposed alternately to the medium and to the gas. When the live slicesare exposed to the potential drug candidate the toxicity of thepotential drug candidate can be determined by observing theeffectiveness of the liver slices to metabolize a compound in thepresence of the potential drug candidate.

In a further embodiment, the system has a mesh at least partiallysurrounding the animal liver slices so as to form a space and to retainthe slices within this space. In this embodiment the mesh isapproximately vertical in the chamber. Additional embodiments have twomeshes at least partially surrounding the liver slices.

The invention also provides methods for evaluating the toxicity of adrug. The methods involve supplying a culture medium, contacting theculture medium with animal liver slices, where the animal liver slicesare positioned securely in a chamber so as to maximize the surface areaof liver slices exposed to the culture medium. The chamber has a plasmainlet and a gas valve, means for selectively supplying and removingplasma in the chamber so that the plasma in the chamber comes intocontact with the liver slices, and is removed from contact with theliver slices, means for supplying a gas to the chamber, and a reservoirfor containing plasma as it enters and exits the chamber. The methodfurther involves contacting the liver slices with a gas mixture ofoxygen and carbon dioxide, exposing the liver slices alternately toplasma and the gas mixture of oxygen and carbon dioxide gas, andexposing the liver slices to the drug to be tested. When the live slicesare exposed to the drug the toxicity of the drug can be determined byobserving the effectiveness of the liver slices to metabolize a compoundin the presence of the drug.

In the embodiments disclosed herein, the compound to be metabolized canbe selected from the group consisting of ammonia and lidocaine.

BRIEF DESCRIPTION OF THE DRAWING

Further particularities and advantages of the invention will becomeclear from the following description of preferred embodiment, withreference to the drawing, in which:

FIG. 1 is a schematic diagram of the system of the present invention;

FIG. 2A is a side sectional view of the liver-slice arrangement of thepresent invention;

FIG. 2B is a perspective view of the liver-slice arrangement of FIG. 2A;

FIG. 3A is a graphical representation of in vitro lidocaine clearancewith continuous and intermittent perfusion using the bioartificial liversystem of the present invention;

FIG. 3B is a graphical representation of in vitro lidocaine clearancewith a 6 hour and a 24 hour run using the bioartificial liver system ofthe present invention;

FIG. 4 is a graphical representation of in vitro DMX concentration witha 6 hour and a 24 hour run using the bioartificial liver system of thepresent invention; and

FIG. 5 is a graphical representation of in vitro ammonia clearance witha 6 hour and a 24 hour run using the bioartificial liver system of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The objective during the pre-clinical drug development stage is for apharmaceutical company to show that the compound is reasonably safe foruse in the next phase, which is small-scale clinical studies. Thecompound's toxic and pharmacologic effects are realized through in vivoand in vitro animal testing. At a minimum, the FDA will ask thepharmaceutical company to: (1) develop a pharmacologic profile of thedrug; (2) determine the acute toxicity of the drug in at least twospecies of animals; and (3) conduct short-term toxicity studies rangingfrom 2 weeks to 3 months, depending on the proposed duration and use ofthe substance in the proposed clinical studies. The process iscomplicated and costly with hundreds and sometimes thousands ofcompounds being tested.

This testing is often performed by an independent third party in orderto rule out any appearance of bias. Every effort is made to ensure thatas few animals as possible are used, and that they are treated humanely.Usually two species of animals, one rodent and one non-rodent are usedbecause a drug may affect one species differently than another. Sincemost drugs are metabolized in the liver, toxicity studies naturallyfocus on the effects on the liver.

The present invention, by using liver slices, is ideally suited to thepre-clinical development process. The number of animals required isminimized, as is the need for subjecting the animal to often stressfuland painful testing procedures.

In accordance with the present invention, there is provided abioartificial liver system for evaluation, detection and testing of drugcandidates, drugs and drug metabolites. The system has a liver-sliceculture apparatus.

In one embodiment of the present invention, the apparatus has a chamberwith a gas valve, and a plurality of animal liver slices positionedsecurely within the chamber so as to maximize the surface area of theliver slices exposed to a medium. There is a means for selectivelysupplying and removing culture medium to the chamber so that the culturemedium in the chamber rises to come into contact with the liver slices.The culture medium rises in the chamber so that the liver slices arecompletely immersed. This means is also able to remove the culturemedium from contact with the liver slices. In additional embodiments,there is also a means for supplying a gas to the top of the chamber sothat the liver slices are exposed alternately to the gas and to theculture medium. Additionally, a reservoir is provided for containing theculture medium as it enters and exits the chamber. The chamber ispreferably thermoregulated. For human liver slices, the temperature ispreferably kept at about 36.5 degrees C. For rodent liver slices, it iskept between about 36 to 38 degrees C. However, pig liver slices arevery sensitive to temperature fluctuation and it must be maintained at38 degrees C., the normal body temperature of pigs.

FIG. 1 is a schematic representation of drug testing system 10 inaccordance with the present invention. From reservoir 12 culture medium13 is introduced into the liver-slice culture apparatus 14. Liver slices15 are arranged between two wire meshes 16 and placed verticallyparallel within the bioreactor. As culture medium is introduced into thebioreactor, the fluid level begins to rise until it comes into contactwith the liver slices, and eventually the liver slices are completelyimmersed.

Oxygenated gas is introduced by gas valve 17 in the top of the chamber.Although the gas valve is shown in the top of the chamber, it is alsocontemplated herein that the gas valve could be on the side or bottom ofthe chamber, provided with an appropriate seal to prevent leakage ofliquid medium. The gas is preferably a mixture of 95% by volume O₂ and5% by volume CO₂, and is supplied at a pressure ranging from 1 to 10 ATMto the chamber through the gas valve and discharged therefrom, whilecontrolling the pressure by a pressure controller (not shown). Asolenoid valve (also not shown) may be coupled with the pressurecontroller to maintain a pre-set gas pressure. Gas sterilizing device18, for example, a syringe filter having a pore size of about 0.22 μm,is preferably installed in gas valve 17 to filter out microbes, therebysterilizing the supply gas to the chamber. Gas check valve 11 with gassterilizing device 18 is positioned on the medium reservoir and servesto equalize the pressure between the reservoir and atmosphere.

Stabilization of the liver slices is an important feature of theinvention. The liver slices are cultured under the supplies of liquidculture medium and an oxygenated gas. The liquid culture medium, or theplasma, is supplied through the reservoir into the chamber and theoxygenated gas is supplied through the top of the chamber. Each issupplied at regular intervals so that each of the liver slices isexposed alternately to the medium and to the gas at an exposure-timeratio ranging from about 1:2 to about 1:4, preferably about 1:2.5 toabout 1:3.5, and most preferably about 1:3. Pump 19 controls the flow ofthe culture medium.

Although plasma is a relatively good medium to maintain cell viability,there are too many unknown factors present and therefore the resultsvary from animal to animal. In the present invention Waymouth MB 752/1medium is preferred over plasma. To prevent central necrosis, the gasmixture described above, 95% O₂ and 5% CO₂, is preferably used. Sincethis mixture may produce free oxygen radicals, which are very toxic toliver cells, a high concentration of glutathione and vitamin E, asoxygen free radical scavengers and anti-oxidants, are added. For use ofthis medium, the formula should be supplemented with 10% complementinactivated Fetal Vovine of Calf Serum and L-Glutamine.

Referring now to the drawing, and more particularly FIGS. 2A and 2B, theliver-slice apparatus of the present invention is shown, as representedby numeral 20. Two stainless steel meshes 21 and 22 are provided, thesize of which can be chosen based on the dimensions of the chamber.These two meshes are preferably arranged in parallel. In a preferredembodiment, the meshes have about a 0.26 mm pore size. Also, in apreferred embodiment, the meshes are pressed to ensure consistentflatness. Between meshes 21 and 22 is a plurality of liver-slices 23arranged in an orderly fashion. The two meshes are positioned on eachside of the liver slices with enough room so as to not crush the liverslices, but also so as to hold them sufficiently so that they do not getwashed away by the plasma. Although FIGS. 2A and 2B show a relativelysmall number of liver slices positioned between the meshes, it is to beunderstood that the efficiency of the apparatus is dependent upon thenumber of liver slices employed. In addition, although two meshes areshown, it is contemplated herein that a single mesh may be used. Thatmesh is formed to surround, at least partially, the liver slices therebyforming a space and to retain them in that space. For example, the meshcould be formed in a suitably dimensioned U-shape.

Liver slices used in the present invention may be obtained from asuitable animal, for example, a rabbit, pig, dog, rodent, or human,depending on the intended use of the apparatus. Also, they may be of anysize or shape suitable for maintaining the viability and essentialfunctions thereof. In the present invention the liver slices arepreferred to have a thickness ranging from about 10 μm to about 2,000μm, and more preferably ranging from about 100 μm to about 500 μm.

The present invention is ideally suited to testing the toxicity andefficiency of a drug. This testing is accomplished by exposing the liverslices to a drug or drug candidate and observing the ability of theliver slices to metabolize a compound, which compound or its metabolitescan be detected. For example, ammonia and lidocaine are common compoundsthat can be metabolized by healthy liver tissue. The following exampleshows this testing.

EXAMPLE 1 In Vitro Performance

The following example illustrates the in vitro performance of the systemusing liver slices and forms the model for the drug testing system ofthe present invention. The example here shows the efficiency of liverslices to metabolize ammonia and lidocaine in the presence of drugcandidate HL100.

The liver converts ammonia to urea, which is excreted into the urine bythe kidneys. In the presence of severe liver disease, ammoniaaccumulates in the blood because of both decreased blood clearance anddecreased ability to form urea. Elevated ammonia levels can be toxic,especially to the brain, and play a role in the development of hepaticencephalopathy. Accordingly, measuring ammonia clearance can assessliver function. More specifically, measuring ammonia clearance providesan indication of the operability of the present invention to metabolizecompounds that may or may not be harmful to the liver.

In addition, lidocaine is a drug that can be converted by the liver froma toxic form into a non-toxic metabolite known as dimethyl xylidine(DMX). The measure of lidocaine clearance is an indication of theperformance of the present invention. By measuring the clearance ofammonia or lidocaine in the presence of the drug candidate, a toxicityprofile for the drug candidate can be generated. If the drug candidateis toxic to liver cells, there will be a build-up of ammonia andlidocaine in these examples. Therefore there is an observable directrelationship between drug candidate toxicity and lidocaine or ammonialevels.

A 3 to 3.5 kg rabbit was euthanized and liver slices obtained. Theslices were approximately 1 cm in diameter with an average weight of 50mg. Approximately 2 grams total were used in this example. The sliceswere then pre-cultured by immersion in approximately 200 ml of WilliamsE media with 10% FCS and drained upon exposure to an oxygenated gas.Each liver slice is exposed alternately to the medium and to the gas atan exposure-time ratio of approximately 1:3.

The gas mixture, approximately 95% oxygen, 5% CO₂ at 1 ATM, wasmaintained in the chamber throughout the study. The gas mixture wasexchanged every twelve minutes. Bolus doses of lidocaine (2 mg) orammonia (20 mg) were injected. The ammonia and DMX were measured bycollecting samples at 0, 5, 15, 30, 60, 90 and 120 minutes, after 6hours and 25 hours of cultivation. The results are summarized in FIGS.3A, 3B, 4 and 5.

FIG. 3A is a graphical representation of in vitro clearance of a 2 mgdose of lidocaine. Continuous perfusion was performed (as indicated bythe diamonds) and intermittent perfusion (time-exposure ratio of 1:3)was also performed (indicated by the circles). Three separate trialswere performed for each. At approximately 30 minutes after lidocaineloading, the level of lidocaine dropped from between 3.2 and 5.8 μg toapproximately 0.9 μg. This level was reduced to approximately 0.5 μg at120 minutes. The results demonstrate that the device of the presentinvention reduced lidocaine levels to non-toxic levels within 30 minuteseven in the presence of drug candidate HL 100. As compared to continuousmedium perfusion, the intermittent perfusion (approximately 1:3)requires less volume of medium while achieving substantially the sameresults. The results show that the drug candidate does not substantiallyimpair the ability of the liver slices to metabolize lidocaine.

FIG. 3B is a graphical representation of in vitro clearance of a 2 mgdose of lidocaine for prior run times of 6 hours and 24 hours. In theseruns, the liver slices were exposed to gas either continuously orintermittently in a ratio of 1:3 for 6 hours and for 24 hours prior tolidocaine loading. Initial readings of lidocaine were between 2 μg and7.8 μg. However, within 30 minutes lidocaine levels reduced toapproximately 0.80 μg for the 6 hour trials and for the continuousperfusion 24 hour trial. Within 60 minutes all trials were showinglidocaine levels between 0.75 μg and 1 μg. Again, the resultsdemonstrate the efficiency of the device to reduce lidocaine levels tonon-toxic levels with intermittent perfusion while exposed to HL 100.

FIG. 4 is a graphical representation of in vitro DMX concentrationbuild-up from a 2 mg lidocaine dose. Initially DMX concentrationremained approximately zero, until approximately 18 minutes. The DMXmetabolites were observed increasing in concentration after 18 minutesand reached approximately maximal values at 60 minutes. However, for the24 hour 1:3 exposure trial, the DMX concentration continued to increaseup to 120 minutes. These results show the ability of the presentinvention to metabolize lidocaine (as indicated by the DMX metaboliteconcentration increasing over time) in the presence of HL 100. There wasno significant difference between the continuous perfusion trial and theintermittent perfusion trial, except for the 24 hour exposure trialmentioned above.

FIG. 5 is a graphical representation of in vitro ammonia clearance of a20 mg loading dose. At approximately 30 minutes maximal ammoniaclearance was observed in all trials. These results demonstrate theability of the present invention to remove ammonia relatively quickly tonon-toxic levels in the presence of drug candidate HL 100. In addition,there was no significant difference between the trials with continuousperfusion and those with intermittent perfusion, thereby indicating thatless medium can be used while still retaining activity and efficiency ofthe device.

While the present invention has been illustrated and described by meansof a specific embodiment, it is to be understood that numerous changesand modifications can be made therein without departing from the scopeof the invention as defined in the accompanying claims.

1. A system for testing a potential drug candidate for toxicity, saidsystem comprising: a liver-slice culture apparatus, the cultureapparatus comprising: a chamber having a medium inlet and a gas valve; aplurality of animal liver slices positioned securely within said chamberso as to maximize the surface area of the liver slices exposed to amedium; and means for selectively supplying and removing a medium in thechamber so that the medium in the chamber comes into contact with theliver slices, and is removed from contact with the liver slices; and areservoir for containing the medium as it enters and exits the chamber,said animal liver slices being cultured in an environment of anoxygenated gas under the supply of the medium at regular intervals sothat said slices are exposed alternatively to the medium and to the gas;wherein when the liver slices are exposed to the potential drugcandidate the toxicity of the potential drug candidate can be determinedby observing the effectiveness of the liver slices to metabolize acompound in the presence of the potential drug candidate.
 2. The systemof claim 1, wherein a mesh at least partially surrounds said animalliver slices so as to form a space and to retain said slices within saidspace, said mesh being approximately vertical in the chamber.
 3. Thesystem of claim 2, wherein two meshes at least partially surround saidliver slices.
 4. The system of claim 1, wherein the liver slices have athickness in the range of about 10 μm to about 2,000 μm.
 5. The systemof claim 1, wherein the liver slices have a thickness in the range ofabout 100 μm to about 500 μm.
 6. The system of claim 1, furthercomprising a means for introducing a gas to the gas valve.
 7. The systemof claim 6, wherein the gas is a mixture of oxygen and carbon dioxide.8. The system of claim 7, wherein the gas-to-plasma exposure time ratioto the animal liver slices is about 1:2 to about 1:4.
 9. The system ofclaim 7, wherein the gas-to-plasma exposure time ratio to the animalliver slices is about 1:3.
 10. The system of claim 1, further comprisingan immunological filter inserted in the gas valve.
 11. The system ofclaim 1, wherein the chamber is sealable.
 12. The system of claim 11,wherein the chamber is thermoregulated.
 13. The system of claim 1,wherein the compound is selected from the group consisting of ammoniaand lidocaine.
 14. A method for evaluating the toxicity of a drug, saidmethod comprising: supplying a culture medium; contacting the culturemedium with animal liver slices, the animal liver slices beingpositioned securely in a chamber so as to maximize the surface area ofliver slices exposed to the culture medium, wherein the chamber has aplasma inlet and a gas valve, means for selectively supplying andremoving plasma in the chamber so that the plasma in the chamber comesinto contact with the liver slices, and is alternately removed fromcontact with the liver slices, means for supplying a gas to the chamber,a reservoir for containing plasma as it enters and exits the chamber,the method further comprising: contacting the liver slices with a gasmixture of oxygen and carbon dioxide; exposing the liver slicesalternatively to plasma and the gas mixture of oxygen and carbon dioxidegas; and exposing the liver slices to the drug to be tested; whereinwhen the liver slices are exposed to the drug the toxicity of the drugcan be determined by observing the effectiveness of the liver slices tometabolize a compound in the presence of the drug.
 15. The method ofclaim 14, wherein a mesh at least partially surrounds said animal liverslices so as to form a space and to retain said slices within saidspace, said mesh being positioned approximately vertical in the chamber.16. The method of claim 15, wherein the compound is selected from thegroup consisting of ammonia and lidocaine.